Human pregnancy sets up an inherent immunological paradox. In a tumor-like process, semi-allogeneic fetal cells of the placenta (cytotrophoblasts;CTBs) invade the uterine lining (decidua) where they reside amongst an unusual subpopulation of maternal immune cells termed decidual granular leukocytes (DGLs), which comprise approximately 40% of the cells in this location. About 30% of DGLs are T cells, dendritic cells and macrophages;the majority (70%) are natural killer (NK) cells. Purified uterine NK (uNK) cells, an unusual CD56brightCD16- population, exhibit reduced but not totally abrogated killing of target cell lines in vitro. Yet, during normal pregnancy, there is no evidence that they lyse CTBs in vivo. Whether uNK cells are capable of lysing primary CTBs in vitro remains an open question that we recently addressed. Specifically, we cultured either DGLs or purified uNK cells with CTBs. The results were dramatic. Consistent with previous findings from our group and other investigators, uNK cells in the context of the total leukocyte population failed to kill CTB targets. However, purified uNK cells quickly surrounded and lysed CTBs, to our knowledge, the first demonstration of this phenomenon. These results suggest a new two-part hypothesis: (1) Specific receptors and ligands mediate uNK lysis of CTBs;and (2) Protection is normally afforded by regulatory cells that reside within the DGL population as a whole. Here we propose experiments that are designed to test this theory. First, we will characterize, in detail, the repertoire of receptors which uNK express that engage MHC class I proteins and other molecules. At the same time, we will profile CTB expression of candidate ligands, information that we will use to expand our list of potential uNK receptors. Second, the results of these experiments will allow us to perform co-culture assays to test the functions of receptors and ligands with expression patterns, which suggest that they are playing important roles. Third, we will identify the regulatory cells in the DGL population as a whole that inhibit uNK lysis of CTBs. Thus, at the conclusion of these experiments, we will have substantially advanced our understanding of the mechanisms that control maternal tolerance of fetal cells during human pregnancy. Beyond human reproduction, this study could impact the fields of NK cell biology and tolerance in general, providing novel strategies for preventing the rejection of transplanted cells and tissues. PUBLIC HEALTH RELEVANCE: The results of the proposed experiments will substantially advance our understanding of the mechanisms that control maternal tolerance of fetal cells during human pregnancy. Beyond human reproduction, this study could impact the fields of NK cell biology and tolerance in general, providing novel strategies for preventing the rejection of transplanted cells and tissues.